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dictyNews Volume 33 Number 04
dictyNews
Electronic Edition
Volume 33, number 4
July 31, 2009
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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A Dictyostelium chalone requires G proteins to regulate proliferation
Deenadayalan Bakthavatsalam, Jonathan M. Choe, Nana E. Hanson,
and Richard H. Gomer
Department of Biochemistry and Cell Biology, Rice University, Houston, TX
BMC Biology, in press
Several studies have shown that organ size, and the proliferation of tumor
metastases, may be regulated by negative feedback loops in which
autocrine secreted factors called chalones inhibit proliferation. However,
very little is known about chalones, and how cells sense them. We
previously identified two secreted proteins, AprA and CfaD, which act as
chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster
than wild type cells, and adding recombinant AprA or CfaD to cells slows
their proliferation.
We show here that cells lacking the G protein components Galpha8,
Galpha9, and Gbeta proliferate faster than wild-type cells despite
secreting normal or high levels of AprA and CfaD. Compared to wild-type
cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only
weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD,
Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate
of increase in mass and protein per nucleus), whereas Galpha9 inhibits
both proliferation and growth. galpha8- cells show normal cell-surface
binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface
rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the
synthesis or processing of the AprA receptor. Like other ligands that
activate G proteins, rAprA induces the binding of [3H]GTP to membranes,
and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced
[3H]GTP binding and the GTPgammaS inhibition of rAprA binding require
Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have
reduced spore viability.
This study shows that Galpha8 and Gbeta are part of the signal
transduction pathway used by AprA to inhibit proliferation but not growth
in Dictyostelium, whereas Galpha9 is part of a different pathway that
regulates both proliferation and growth, and that a chalone signal
transduction pathway uses G proteins.
Submitted by Richard Gomer [richard@rice.edu]
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Dictyostelium discoideum CenB Is a Bona Fide Centrin Essential for
Nuclear Architecture and Centrosome Stability
Sebastian Mana-Capelli,1 Ralph Gräf,2 and Denis A. Larochelle1*
Department of Biology, Clark University, 950 Main Street, Worcester,
Massachusetts 01610,1
Institut für Biochemie und Biologie, Zellbiologie, Universität Potsdam,
Karl-Liebknecht-Strasse 24-25, Haus 26, 14476 Potsdam-Golm, Germany2
Eukaryotic Cell, in press
Centrins are a family of proteins within the calcium-binding EF-hand
superfamily. In addition to their archetypical role at the MTOC, centrins
have acquired multiple functionalities throughout the course of evolution.
For example, centrins have been linked to different nuclear activities,
including mRNA export and DNA repair. Dictyostelium discoideum centrin B
is a divergent member of the centrin family. At the amino acid level,
DdCenB shows 51% identity with its closest relative and only paralog,
DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a
well-supported monophyletic and divergent group within the centrin
family of proteins. Interestingly, fluorescently tagged versions of
DdCenB were not found at the centrosome (in whole cells or in isolated
centrosomes). Instead, DdCenB localized to the nuclei of interphase cells.
This localization disappeared as the cells entered mitosis, although
Dictyostelium cells undergo a closed mitosis in which the nuclear envelope
(NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear
architecture, characterized by enlarged and deformed nuclei and loss of
proper centrosome-nucleus anchoring (observed as NE protrusions). At the
centrosome, loss of DdCenB resulted in defects in the organization and
morphology of the MTOC and supernumerary centrosomes and centrosome-related
bodies. The multiple defects that the loss of DdCenB generated at the
centrosome can be explained by its atypical division cycle, transitioning
into the NE as it divides at mitosis. On the basis of these findings, we
propose that DdCenB is required at interphase to maintain proper nuclear
architecture, and before delocalizing from the nucleus, DdCenB is part of
the centrosome duplication machinery.
Submitted by Denis Larochelle [dlarochelle@clarku.edu]
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Dynamic localization of G proteins in Dictyostelium discoideum
Carrie A. Elzie, Jennifer Colby, Morgan A. Sammons, and Chris Janetopoulos
Journal of Cell Science,in press
Extracellular stimuli exert their effects on eukaryotic cells via
serpentine G-protein-coupled receptors and mediate a vast number of
physiological responses. Activated receptors stimulate heterotrimeric
G-proteins, consisting of three subunits, alpha, beta and gamma. In
Dictyostelium discoideum, cAMP binds to the cAMP receptor cAR1, which
is coupled to the heterotrimer containing the G{alpha}2 subunit. These
studies provide in vivo evidence as to how receptors influence the
localization of the G-protein complex prior to and after ligand binding.
Previous work has shown that the state of the heterotrimer could be
monitored by changes in fluorescence (or Förster) resonance energy
transfer (FRET) between the alpha2- and beta-subunits of D. discoideum.
We now report the kinetics of G-protein activation as a loss of FRET
prior to and after cAMP addition by using total internal reflection
fluorescence microscopy (TIRFM). We also performed photobleaching
experiments to measure G-protein recovery times. Our data show that
inactive and active G-proteins cycle between the cytosol and plasma
membrane. These data suggest that cAR1 activation slows the membrane
dissociation ('off') rate of the alpha2 subunit, while simultaneously
promoting beta/gamma-subunit dissociation.
Submitted by: Chris Janetopoulos [c.janetopoulos@Vanderbilt.Edu]
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[End dictyNews, volume 33, number 4]
